Skeletal muscle insulin resistance is nearly universal in type 2 diabetes (T2DM), and of the known diabetogenic risk factors, insulin resistance has one of the greatest predictive values for the development of T2DM. Therefore, interventions designed to reverse skeletal muscle insulin resistance are likely to be effective in preventing and treating this disease. Toll-like receptor (TLR) 4 and inflammatory pathways downstream of this receptor [IKK/IkB/NFkB and c-jun n-terminal kinase (JNK)] have been recently implicated in the pathogenesis of lipid-induced insulin resistance. However, it is not known whether insulin resistant subjects have abnormal TLR4 signaling in the skeletal muscle. The goal of this study is to examine whether TLR4 is implicated in the mechanism underlying skeletal muscle insulin resistance in human subjects. Using the insulin clamp technique with muscle biopsies, and a primary human muscle cell culture system, we plan to test the hypothesis that elevated TLR4 signaling plays an important role in the pathogenesis of lipid-mediated insulin resistance. The following Aims are proposed: 1) Determine whether insulin resistant subjects have abnormal TLR4 expression/content and TLR4-driven signaling in skeletal muscle and whether this predicts abnormalities in insulin signaling and insulin sensitivity;2) Determine whether an experimental elevation in circulating free fatty acids (FFAs) within a physiologic range, increases TLR4 expression/content and stimulates TLR4-driven signaling in muscle from lean normal glucose tolerant (insulin-sensitive) subjects;3) Determine whether the reduction of FFAs, brought about the antilipolytic drug Acipimox, improves TLR4 signaling in muscle from insulin resistant (obese and T2DM) subjects;4) Determine whether TLR4 mediates FFA-induced insulin resistance in human myotubes. These studies will yield new insights into the molecular mechanisms responsible for lipid-induced insulin resistance in muscle from human subjects.The skeletal muscle from subjects with obesity and type 2 diabetes is resistant to the effect of insulin, a hormone, which helps maintain glucose levels within a normal range. However, the cause for the insulin resistance in muscle is not well known. Recent studies done in animals suggest that increased tissue levels of a protein called TLR4 may play a role in the insulin resistance present in subjects with obesity and type 2 diabetes. In this project we plan to examine whether this protein (TLR4) is involved in the molecular mechanisms responsible for insulin resistance in muscle from human subjects.